In vitro cytotoxic and antimicrobial effects of a novel peroxysesquiterpene glucoside from the rhizomes of Cyperus rotundus L (Cyperaceae)

Purpose: To study the antimicrobial and cytotoxic potential of a novel 3,9-peroxsesquiterpene-15-Oglucoside from Cyperus rotundus rhizomes, against HeLa cell line and selected strains of microorganisms. Methods: The rhizomes were macerated with methanol and fractionated with n-hexane, chloroform, ethyl acetate and butanol. High-performance liquid chromatography (HPLC) was performed together with chemical analysis of the fractions. The 3,9-peroxysesquiterpene-15-O-glucoside was purified through column chromatography of the ethyl acetate fraction, and its purity was determined via reversephase HPLC. Structural elucidation was done with Infrared (IR), proton-nuclear magnetic resonance (HNMR), carbon-13 nuclear magnetic resonance (13C NMR), and mass spectrometry (MS) spectroscopic analyses. Results: The isolated compound exhibited bactericidal and fungicidal activities against S. aureus and C. albicans at concentration, respectively, in the range of 32 – 100 μg/mL, while MTT assay results showed the cytotoxicity of the compound against eukaryotic (HeLa) cell line (IC50, 88.32 μg/mL). Conclusion: The isolated metabolite from the methanol extract of C. rotundus rhizome exhibits bactericidal, fungicidal, and cytotoxic potential. However, further studies are required to ascertain its suitability for use as a therapeutic agent. Keywords: Terpenoids, Column chromatography, Spectroscopy, Antimicrobial, Cytotoxicity, Cyperus rotundu

Synthetic Biotechnology to Study and Engineer Ribosomal Bottromycin Biosynthesis

SummaryBottromycins represent a promising class of antibiotics binding to the therapeutically unexploited A-site of the bacterial ribosome. By inhibiting translation they are active against clinically important pathogens, such as vancomycin-resistant Enterococci. Structurally, bottromycins are heavily modified peptides exhibiting various unusual biosynthetic features. To set the stage for compound modification and yield optimization, we identified the biosynthetic gene cluster, used synthetic biotechnology approaches to establish and improve heterologous production, and generated analogs by pathway genetic engineering. We unambiguously identified three radical SAM methyltransferase-encoding genes required for various methylations at unactivated carbons yielding tert-butyl valine, methyl-proline, and β-methyl-phenylalanine residues, plus a gene involved in aspartate methyl-ester formation. Evidence for the formation of the exo-thiazole unit and for a macrocyclodehydration mechanism leading to amidine ring formation is provided

Biosynthesis of Crocacin Involves an Unusual Hydrolytic Release Domain Showing Similarity to Condensation Domains

SummaryThe crocacins are potent antifungal and cytotoxic natural compounds from myxobacteria of the genus Chondromyces. Although total synthesis approaches have been reported, the molecular and biochemical basis guiding the formation of the linear crocacin scaffold has remained unknown. Along with the identification and functional analysis of the crocacin biosynthetic gene cluster from Chondromyces crocatus Cm c5, we here present the identification and biochemical characterization of an unusual chain termination domain homologous to condensation domains responsible for hydrolytic release of the product from the assembly line. In particular, gene inactivation studies and in vitro experiments using the heterologously produced domain CroK-C2 confirm this surprising role giving rise to the linear carboxylic acid. Additionally, we determined the kinetic parameters of CroK-C2 by monitoring hydrolytic cleavage of the substrate mimic N-acetylcysteaminyl-crocacin B using an innovative high-performance liquid chromatography mass spectrometry-based assay

Molekulare Untersuchungen zum Einfluß von Umweltfaktoren und subinhibitorischen Antibiotikakonzentrationen auf die Biofilmbildung in Staphylococcus epidermidis

Biofilm production is an important step in the pathogenesis of S. epidermidis polymer-associated infections and depends on the expression of the icaADBC operon leading to the synthesis of a polysaccharide intercellular adhesin (PIA). The PIA represents a sugar polymer consisting of ß-1,6 linked N-acetyl glucosaminoglycans and mediates the intercellular adherence of the bacteria to each other and the accumulation of a multilayered biofilm. Epidemiological and experimental studies strongly suggest that PIA-production and subsequently biofilm formation contributes significantly to the virulence of specific S. epidermidis strains. This work aimed on the investigation of external factors regulating the ica expression in S. epidermidis. For this purpose, a reporter gene fusion between the ica promoter and the beta-galactosidase gene lacZ from E. coli was constructed and integrated into the chromosome of an ica positive S. epidermidis clinical isolate. The reporter gene fusion was used to investigate the influence of external factors and of sub-MICs of different antibiotics on the ica expression. It was shown that the S. epidermidis biofilm formation is growth phase dependent with a maximum expression in the late logarithmic and early stationary growth phase. The optimal expression was recorded at 42 °C at a neutral pH ranging from 7.0 to 7.5. The glucose content of the medium was found to be essential for biofilm formation, since concentrations of 1.5 to 2 per cent glucose induced the ica expression. In addition, external stress factors as high osmolarity (mediated by 3 to 5 per cent sodium chloride), and sub-lethal concentrations of detergents, ethanol, hydrogene peroxide, and urea significantly enhanced the biofilm production. Subinhibitory concentrations of tetracyline, the semisynthetic streptogramin quinupristin/dalfopristin and the streptogramin growth promoter virginiamycin were found to enhance the ica expression 8 to 11-fold, respectively, whereas penicillin, oxacillin, gentamicin, clindamycin, vancomycin, teicoplanin, ofloxacin, and chloramphenicol had no effects. A weak induction was recorded for sub-MICs of erythromycin. Both quinupristin/ dalfopristin and tetracyline exhibited a strong postexposure effect on the S. epidermidis ica expression, respectively, even when the substances were immediately removed from the growth medium. The results were confirmed by Northern blot analysis of the ica transcription and quantitative analysis of biofilm formation in a colorimetric assay. Expression of the icaprom::lacZ reporter gene plasmid in Bacillus subtilis and S. epidermidis revealed that the ica induction by sub-MICs of streptogramins and tetracycline might depend on unidentified regulatory elements which are specific for the staphylococcal cell. In contrast, the activation by external stress signals seems to be mediated by factors which are present both in Staphylococci and in Bacillus subtilis. Construction and analysis of an agr-mutant in a biofilm-forming S. epidermidis strain excluded the possibility that the Agr-quorum-sensing system significantly contributes to the ica expression in the stationary growth phase. However, clear evidence was provided that in S. aureus the ica transcription depends on the expression of the alternative transcription factor sigmaB, which represents a global regulator of the stress response in S. aureus as well as in B. subtilis. For this purpose, a sigB knockout mutant had been constructed in a biofilm-forming S. aureus. This mutant showed a markedly decrease of the ica transcription and biofilm-production, whereas a complement strain carrying the sigB gene on an expression vector completely restored the biofilm-forming phenotype of the S. aureus wild type. Southern blot analysis indicated that the the sigB gene is also present in S. epidermidis and Northern analyses of the sigB and the ica transcription revealed that both genes are activated under identical conditions (i. e. in the stationary growth phase and by external stress factors) suggesting a similar regulatory pathway as in S. aureus. However, since neither in S. aureus nor in S. epidermidis the ica promoter has obvious similiarities to known SigB-dependent promotoer sequences it is tempting to speculate that the ica activation is not directely mediated by SigB, but might be indirectely controlled by other SigB-dependent regulatory elements which remain to be elucidated.Die Ausbildung von Biofilmen ist ein wichtiger Schritt in der Pathogenense von Polymer-assoziierten Infektionen durch S. epidermidis. Sie hängt von der Expression des icaADBC-Operons ab, das die Syntheseenzyme für die Produktion eines extrazellulären Polysaccharids (PIA) kodiert. PIA steht für Polysaccharid-Interzelluläres-Adhäsin. Diese Substanz stellt ein beta-1,6 verknüpftes N-Acetyl-Glukosaminoglycan dar, das den Bakterien extrazellulär aufliegt, die Adhärenz der Zellen untereinander vermittelt und so die Akkumulation eines Biofilms wesentlich fördert. Epidemiologische und experimentelle Studien haben gezeigt, daß die PIA-Produktion und damit die Biofilmbildung wesentlich zur Virulenz bestimmter S. epidermidis Stämme beiträgt. Die vorliegende Arbeit zielte auf die Untersuchung äußerer Faktoren ab, die die PIA-Expression in S. epidermidis regulieren. Dazu wurde eine Genfusion zwischen dem ica-Promotor und dem beta-Galaktosidasegen aus E. coli hergestellt und stabil in das Chromosom eines ica-positiven S. epidermidis Klinikisolates integriert. Das Reportergenkonstrukt wurde benutzt, um den Einfluß verschiedener Umweltfaktoren und die Wirkung subinhibitorischer Antibiotikadosen auf die Expression des ica-Operons zu bestimmen. Es konnte gezeigt werden, daß die ica-Expression wachstumsphasenabhängig ist und ihr Maximum in der späten logarhitmischen und frühen stationären Phase erreicht. Die optimale Expressionstemperatur liegt bei 42 °C und einem neutralen pH-Wert in einem Bereich zwischen 7,0 und 7,5. Die Biofilmbildung hängt stark vom Glukosegehalt des Wachstumsmediums ab. So wurde gezeigt, daß 1,5 bis 2 Prozent im Kulturmedium die ica-Expression in der logarhitmischen Phase induzieren. Externe Stressfaktoren wie erhöhte Osmolarität durch 3 bis 5 Prozent NaCl oder sublethale Dosen von Detergenzien, Äthanol, Wasserstoffperoxid und Harnstoff begünstigen ebenfalls die ica-Expression und damit die Biofilmbildung. Subinhibitorische Dosen von Tetrazyklin, des Streptogramingemisches Quinupristin/Dalfopristin (Synercid?) sowie des Streptogramin-Leistungsförderers Virginiamycin führten zu einer Erhöhung der ica-Expression um das 8 bis 11-fache. Eine kurzzeitige Exposition der Bakterien zu hohen Konzentrationen dieser Antibiotika führte ebenfalls zu einer langanhaltenden Induktion der Biofilmexpression, auch wenn die Stoffe aus dem Wachstumsmedium sofort wieder entfernt worden waren. Andere Antibiotika wie Penicillin, Oxacillin, Gentamicin, Clindamycin, Vancomycin, Teicoplanin und Chloramphenicol zeigten keinerlei Effekt auf die ica-Expression. Eine schwache Induktion wurde dagegen für Erythromycin verzeichnet. Die Daten wurden durch Northernblotanalysen der ica–Transkription und durch einen quantitativen Biofilmassay in Polystyrol-Mikrotiterplatten bestätigt. Die Propagierung des Reportergenkonstruktes als Plasmid sowohl in einem B. subtili- Hintergrund als auch in einem ica-negativen S. epidermidis-Stamm ergab, daß die Induktion des ica-Operons durch niedrige Tetrazyklin- oder Streptograminkonzentrationen möglicherweise von spezifischen Regulatoren abhängt, die nur in Staphylokokken vorkommen. Im Gegensatz dazu ist die Induktion durch externe Sressfaktoren sowohl in B. subtilis als auch in S. epidermidis möglich. Durch die Herstellung und Untersuchung einer agr-Mutante in einem Biofilm-bildenden S. epidermidis-Stamm konnte ausgeschlossen werden, daß das Agr-Quorum-Sensing-System einen wesentlichen Einfluß auf die ica-Expression in der stationären Phase ausübt. Dagegen wurde klar gezeigt, daß die ica-Transkription in S. aureus von der Expression des alternativen Transkriptionsfaktors SigmaB abhängt, der einen globalen Regulator für die Stressantwort in Staphylokokken und in B. subtilis darstellt. Zu diesem Zweck war eine sigB-Deletionsmutante in einem Biofilm-bildenden S. aureus-Stamm hergestellt worden. Diese Mutante zeigte eine deutlich verminderte ica Transkription und Biofilmbildung. Dagegen wies die Komplementante, die das sigB-Gen auf einem Expressionsvektor trug, wieder alle Merkmale des Biofilm-bildenden S. aureus Wildtyps auf. Southernblot-Analysen zeigten, daß das sigB-Gen auch in S. epidermidis nachweisbar ist und unter denselben Bedingungen (d. h. in der stationären Phase und durch externen Stress) wie das ica Gen transkribiert wird. Das läßt vermuten, daß die Regulation des ica-Operons in S. epidermidis ähnlich abläuft wie in S. aureus und durch SigB beeinflußt wird. Da jedoch weder in S. aureus noch in S. epidermidis der ica-Promotor Ähnlichkeiten zu den bekannten SigB-abhängigen Promotor-Strukturen aufweist, ist anzunehemen, daß dieses Operon nicht direkt durch diesen alternativen Transkriptionsfaktor kontrolliert wird. Wahrscheinlich existieren noch weitere regulatorische Elemente, die hieran beteiligt sind und die noch zu identifizieren sind

On Column Binding a Real-Time Biosensor for β-lactam Antibiotics Quantification

This work aimed to develop accurate, quick, and practical tools for the detection of residues of penicillin G antibiotic in biological and non-biological samples. The assays were developed based on the binding mechanism of β-lactam to penicillin-binding proteins; samples of different concentrations of penicillin G were incubated with in vitro expressed 6X-Histidine-tagged soluble penicillin-binding protein (PBP2x*) of Streptococcus pneumoniae (S. pneumoniae), whereby penicillin G in samples specifically binds to PBP2x*. The fluorescent-labeled β-lactam analogue Bocillin FL was used as a competent substrate, and two different routes estimated the amounts of the penicillin G. The first route was established based on the differences in the concentration of non-bounded Bocillin FL molecules within the reactions while using a real-time polymerase chain reaction (PCR)-based method for fluorescence detection. The second route depended on the amount of the relative intensity of Bocillin FL bounded to Soluble PBP-2x*, being run on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-page), visualized by a ChemiDoc-It®2 Imager, and quantified based on the fluorescence affinity of the competent substrate. While both of the methods gave a broad range of linearity and high sensitivity, the on column based real-time method is fast, non-time consuming, and highly sensitive. The method identified traces of antibiotic in the range 0.01–0.2 nM in addition to higher accuracy in comparison to the SDS-based detection method, while the sensitivity of the SDS-based method ranged between 0.015 and 2 µM). Thus, the on column based real time assay is a fast novel method, which was developed for the first time based on the binding inhibition of a fluorescence competitor material and it can be adapted to screen traces of penicillin G in any biological and environmental samples

Comparative Analysis of the Fatty Acid Pattern of Silybum marianum with Nigella sativa by Gas Chromatography-Mass Spectrometry

Many studied were conducted to evaluate the antihepatotoxic and antioxidant activities of Silybum marianum and proved these actions. The Naturally grown seed in Iraqi-Kurdistan Region also were studied for its chemical contents and biological activities. Vegetable oils occur in various plant parts mainly concentrated in the seeds. In this study comparison was made between the fatty acid patterns of two plant seeds, Silybum marianum and Nigella sativa. Seed sample of Silybum marianum and Nigella sativa were exposed for extraction and isolation of the fatty acid contents using two different solvents (petroleum ether and n-hexane) at 60-80oC using soxhlet apparatus and the oily extract were purified and analysed by GC-MS triplet system. Result showed that extraction with petroleum ether yielded 23% and 24% of total weight of the seeds of Silybum marianum and Nigella sativa respectively and comparable results with n-hexane extract yielded 17% and 22% of total seed weight of Silybum marianum and Nigella sativa respectively showing significant differences in the fatty acids patterns of both plants under research. Key words: Fatty acids, GC-MS, Silybum marianum, natural products, cardiovascular diseases.

A Single-Center Study of Viral Respiratory Tract Infections in Hospitalized Children From the Kurdistan Region of Iraq

Viral respiratory infections are among the most common causes of disease in humans, particularly in young children, and remain a major public health problem worldwide. For many geographic regions, there is limited epidemiological information on the main causative agents of these diseases. In this article, we investigated, in a prospective study, the viral agents leading to acute respiratory disease in children younger than 15 years of age who were admitted to the pediatric emergency unit of a major teaching hospital in Erbil City, capital of the Kurdistan region, Iraq. Nasopharyngeal samples obtained from 269 hospitalized children were analyzed for viral respiratory pathogens using the xTAG Respiratory Virus Panel Fast assay, and the data were correlated with the clinical and demographic information available for these patients. One or more respiratory virus(es) were detected in 203 out of 269 (75.5%) samples. The most frequent viruses were enterovirus/rhinovirus (n = 88; 32.7%), respiratory syncytial virus (n = 55; 20.4%), and human metapneumovirus (n = 36; 13.4%). In 42 samples (15.6%), coinfections with 2 or more respiratory viruses were detected, with enterovirus/rhinovirus, respiratory syncytial virus, human metapneumovirus, and adenovirus being identified as the most common agents in viral coinfections in these patients